CN104744568A - P substance peptide probe capable of specific recognition of neurokinin-1 receptor protein, and preparation and application thereof - Google Patents
P substance peptide probe capable of specific recognition of neurokinin-1 receptor protein, and preparation and application thereof Download PDFInfo
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- CN104744568A CN104744568A CN201310726653.3A CN201310726653A CN104744568A CN 104744568 A CN104744568 A CN 104744568A CN 201310726653 A CN201310726653 A CN 201310726653A CN 104744568 A CN104744568 A CN 104744568A
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Abstract
The invention provides a P substance peptide probe capable of specific recognition of a neurokinin-1 receptor protein. the P substance peptide probe is a polypeptide dimer or a polypeptide polymer, the monomer is formed by a P substance amino acid sequence MLGFFQQPKPR, a bifunctional chelating group, a fluorescence group and a terpyridyl group connected through lysine. The novel peptide probe is used for the preparation of a polypeptide dimer and polypeptide polymer with function of diagnosing multi tumors. The polypeptide dimer and polypeptide polymer marks metal Gd<3+> to a polypeptide molecule by the bifunctional chelating group; the marked P substance peptide probe and the NK-1 receptor protein specifically bind and concentrate to the tumor site in vivo; and magnetic imaging and optical imaging technology are utilized for detection of the neurokinin-1 receptor protein to improve the sensitivity of the detection.
Description
Technical field
The present invention relates to the pharmaceutical field of diagnosing tumor, what be specifically related to is Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe to the identification of neurokinine-1 receptor protein-specific and preparation and application thereof, this Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe is novel polypeptide dimer and polypeptide polymer, detects tumour by neurokinine-1 (NK-1) receptor protein in specific binding tumour.
Background technology
Lung cancer is one of modal malignant tumour, in China's urban population Death Cause for Malignant Tumors, lung cancer ranks first, the M & M of countries in the world lung cancer had the trend obviously increased in recent years, but its result for the treatment of does not significantly improve in nearly 10, total curative ratio is about 10%.Current early diagnosis lacks specific index and method, and the inspection by stages of some effects diagnostic is difficult to universal enforcement, and patients with lung cancer is early stage without specific symptom, therefore mostly is middle and advanced stage time medical.
Targeted therapy, namely on cellular and molecular level, for clear and definite carcinogenic site, (this site can be a protein molecular of inside tumor cells, also can be a gene fragment), design corresponding medicine, medicine enters in body can be selected carcinogenic site to combine to have an effect specifically, makes tumor cell specific dead, and the normal tissue cell that can not involve around tumour, this methods for the treatment of becomes one of Main way studied from now on gradually.Tumor tissues is a special microenvironment, wherein comprises and does not much have in normal cell or albumen that content is very low, as neurokinine-1 receptor albumen.This albumen is expressed in the various kinds of cell of lung cancer, as endotheliocyte, epithelial cell, smooth muscle cell, and promotes growth and the transfer of these cells.Therefore this albumen can be used as a target of detection of lung cancer, carry out diagnosing by detecting nk 1 receptor albumen.
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 belongs to tachykinin family, and its aminoacid sequence is MLGFFQQPKPR, participates in various physiological processes, and research in recent years finds, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, as a kind of neurotransmitter, can pass through autocrine or paracrine, be combined with its acceptor NK-1.This shows, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 is the targeting molecule for pulmonary cancer diagnosis of an a lot of prospect, and likely also has potential using value to other malignant tumours.In fact, nk 1 receptor albumen not only has expression in lung cancer, is also present in ovarian cancer, mammary cancer, prostate cancer, neurospongioma, colorectal carcinoma, therefore the probe containing Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 can the above kinds of tumors of specific detection.But up to the present, do not report and utilize Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 probe to detect the document of nk 1 receptor albumen by magnetic imaging and the difunctional imaging of fluorescence imaging, thus the present invention propose first utilize Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe detect tumour.Its advantage is by supramolecular thought improvement on synthesis dimer and polypeptide polymer, improves by magnetic imaging and the difunctional imaging of fluorescence imaging two kinds of means the susceptibility detecting neurokinine-1 receptor albumen.
Summary of the invention
The object of the present invention is to provide a kind of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe to the identification of neurokinine-1 receptor protein-specific and preparation and application thereof, this polypeptide probe be a class novel for the preparation of polypeptide dimer and the polypeptide polymer with kinds of tumors diagnostic effect, this polypeptide dimer and polypeptide polymer by difunctional chelation group by Metal Gd
3+mark is on peptide molecule, and the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe in vivo through mark gathers tumor locus with nk 1 receptor protein-specific in conjunction with dense, utilizes magnetic imaging and optical image technology to detect neurokinine-1 receptor albumen, to improve detection sensitivity.
The object of the invention is to be achieved through the following technical solutions:
The invention provides the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe to the identification of neurokinine-1 receptor protein-specific, it is characterized in that: described Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe is polypeptide dimer or polypeptide polymer, monomer whose is formed by connecting by Methionin by one section of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 aminoacid sequence MLGFFQQPKPR, difunctional chelation group, fluorophor and terpyridine moieties.
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, preferably, the single amino acid sequence of described polypeptide dimer is: MLGFFQQPKPRKKKKKK (tpy) KK (Dye) DOTA.
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, preferably, the single amino acid sequence of described polypeptide polymer is: MLGFFQQPKPRKKKKKK (tpy) K (tpy) KK (Dye) DOTA.
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, adding a series of Methionin one after Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 aminoacid sequence is to strengthen the water-soluble of peptide chain, and two is to try one's best away from Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 sequence above, avoiding adding the structure and function that macoradical may change Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2.Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, between 17 and 19, add a Methionin is sterically hindered for reducing, and described two groups order can be exchanged.
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, preferably, the polymerization degree of described polypeptide polymer is 3-5.
Dimeric peptide chain of the present invention there is a terpyridine moieties, can only dimer be formed; There are two terpyridine moieties in polypeptide polymer monomer of the present invention, can be formed by covalent linkage by a lot of monomers in theory, but in this application, because the reason such as sterically hindered causes the polymerization degree of described polypeptide polymer at 3-5.
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, preferably, described difunctional chelation group is DOTA, and described DOTA is tri-tert DOTA.
Described difunctional chelation group can chelated mineral Gd
3+, F
19, Mn
2+.
The preferred chelated mineral Gd of difunctional chelation group of the present invention
3+, at present for the mainly Gd of contrast medium clinically
3+title complex contrast medium.
Described difunctional chelation group, because polypeptide does not have the functional group directly forming stable bond with metal, so the method for solution be modify on polypeptide some can with the group of metal ion mortise.The difunctional chelation group of the present invention has the group be easily combined with polypeptide amino acid residue, have again can with the functional group of metal ion stable complexation, by its effect, the mixture of magnetic imaging metal one bifunctional chelating agent one antibody (polypeptide) can be formed.Use DOTA as bifunctional chelating agent in the present invention, after this chelant ties metal, there is satisfactory stability.
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, preferably, described terpyridine moieties is selected from 4'-(4-carboxymethoxyl phenyl)-2,2':6', 2''-terpyridyl), 4'-(4-carboxyl phenyl)-2,2':6', 2''-terpyridyls, 4'-carboxyl-2, one in 2':6', 2''-terpyridyl.
4'-(4-carboxymethoxyl phenyl)-2,2':6', 2''-terpyridine structure formulas:
4'-(4-carboxyl phenyl)-2,2':6', 2''-terpyridine structure formula:
4'-carboxyl-2,2':6', 2''-terpyridine structure formula:
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, preferably, described fluorophor sends strong fluorescence at 610nm, realizes fluoroscopic examination.
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, preferably, described fluorophor is hemicyanine dye.
Described hemicyanine dye have molar extinction coefficient large, be easy to synthesis and the advantage such as purifying, suitable fluorescence quantum yield, be widely used in many fields.Hemicyanine dye of the present invention can with a lot of biomolecules, and cause the movement of spectrum, the change of intensity.The structure of hemicyanine dye is obtained by cyanine dyes, and cyanine dyes structure is as figure below, and it is the integral part of heteronucleus that 2 nitrogen-atoms of cyanine dyes are, if in Molecule of Cyanine Dyes only 1 nitrogen-atoms be the integral part of heteronucleus; be called half cyanines.
Specifically, fluorophor of the present invention is preferably from 2-[4-(N, N-bis-propyloic) amino] styryl-1,3,3-Three methyl Benzene diindyl iodine is called for short BIDC1,2-[4-(N, N-bis-propyloic) amino] styryl-1-butyl-3,3-dimethyl benzene diindyl iodine (BIDC2) and 2-[4-(N, N-bis-propyloic) is amino] styryl-1-octyl group-3,3-dimethyl benzene diindyl iodine (BIDC3).
The object that the present invention chooses fluorescence dye can send fluorescence to be convenient to detect, as long as other dyestuffs that can realize this function can under specific wavelength excites.
The present invention also provides a kind of method preparing Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe to the identification of neurokinine-1 receptor protein-specific, comprises the following steps:
The preparation of a, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 sequence
(1) synthesis of microwave Peptide synthesizer is used, Rink amide resins and Fmoc (fluorenylmethyloxycarbonyl) methionine(Met) are placed in reaction flask, add organic bases and couplant, at DMF(N, dinethylformamide) in solution, after reacting 30min under microwave condition, then remove Fmoc blocking group, obtain the resin with methionine(Met) coupling;
(2) repeating step (1) successively, wherein raw material amino acid replaces with Fmoc leucine successively, Fmoc glycine, Fmoc phenylalanine, Fmoc phenylalanine, the related Fmoc glutamine having protecting group in side, the related Fmoc glutamine having protecting group in side, Fmoc proline(Pro), the related Fmoc Methionin having protecting group in side, Fmoc proline(Pro), the related Fmoc arginine having protecting group in side, the related Fmoc Methionin having protecting group in side, the related Fmoc Methionin having protecting group in side, the related Fmoc Methionin having protecting group in side, the related Fmoc Methionin having protecting group in side, the related Fmoc Methionin having protecting group in side, finally obtain the resin with above-mentioned amino acid successively coupling,
(3) step (2) is obtained and the related Fmoc Methionin having Alloc protecting group of the resin of above-mentioned amino acid successively coupling and side, be placed in reaction flask, add organic bases and couplant, in DMF solution, after reacting 30min under microwave condition, obtain further with the resin of the Fmoc Methionin coupling with Alloc protecting group;
The preparation of b, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer
(1) in the resin of step a gained and amino acid successively coupling, Pd (Ph is added
3p)
4(tetra-triphenylphosphine palladium) and PhSiH
3(phenylsilane), reacts 10 minutes, removes the Allco(allyloxycarbonyl on lysine side-chain in DMF) protecting group;
(2) step (1) products therefrom and terpyridyl are placed in reaction flask, add organic bases and couplant, in DMF solution after reaction, then remove Fmoc blocking group;
(3) then under the condition of organic bases, couplant and DMF solution, successively with side the related Boc of having protecting group Fmoc Methionin, side is related has the Fmoc Methionin of Allco protecting group to react;
(4) repeating step (1), (2), its (2) Raw replaces with hemicyanine dye, DOTA, finally obtain being connected with terpyridyl, hemicyanine dye, DOTA with the resin of amino acid successively coupling;
(5) resin of cutting step (4) gained and amino acid coupling successively, obtains Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer monomer;
(6) Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer monomer is dissolved in ultrapure water, adds GdCl
3, after stirred overnight at room temperature, filter, filtrate is through dialysis, and freeze-drying obtains Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer monomer of mark;
(7) by Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer monomer of mark is dissolved in ultrapure water, adds FeCl
2, after stirred overnight at room temperature, dialysis, freeze-drying obtains Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer of mark;
The preparation of c, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer
(1) by the resin of step a gained and amino acid successively coupling, remove Fmoc blocking group, under organic bases, couplant and DMF solution existent condition, have the Fmoc Methionin of protecting group Allco to react with side is related, Fmoc Methionin is coupled on peptide chain;
(2) in step (1) products obtained therefrom, Pd (Ph is added
3p)
4and PhSiH
3, react 10 minutes in DMF, remove the Allco protecting group on two Fmoc lysine side-chains;
(3) step (2) products obtained therefrom and terpyridyl are placed in reaction flask, add organic bases and couplant, in DMF solution after reaction, remove Fmoc blocking group;
(4) repeating step (3), its Raw replaces with Fmoc Methionin, the related Fmoc Methionin having Allco protecting group in side of the side related Boc of having protecting group successively;
(5) repeating step (2), (3), wherein step (3) Raw replaces with hemicyanine dye, DOTA successively, finally obtain being connected with terpyridyl, hemicyanine dye, DOTA with the resin of amino acid successively coupling;
(6) resin of cutting step (5) gained and amino acid coupling successively, obtains Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer monomer;
(7) Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer monomer is dissolved in ultrapure water, adds GdCl
3, after stirred overnight at room temperature, filter, filtrate is through dialysis, and freeze-drying obtains Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer monomer of mark;
(8) by Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer monomer of mark is dissolved in ultrapure water, adds FeCl
2, after stirred overnight at room temperature, dialysis, freeze-drying obtains Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer of mark.
The preparation method of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention; preferably, relatedly there is the Fmoc glutamine of protecting group described side, side is related the Fmoc Methionin of protecting group, related Fmoc glutamine, the side related Boc(of the having tertbutyloxycarbonyl having the Fmoc arginine of protecting group to be followed successively by the side related Trt of having protecting group in side) Fmoc Methionin, the related Fmoc arginine having Pbf protecting group in side of protecting group.
The preparation method of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, preferably, described organic bases is DIPEA (DIPEA), and described couplant is PyBOP (phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl).
In cutting step of the present invention, remove amino acid side simultaneously and connect protecting group.
The preparation method of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, the single amino acid sequence of described polypeptide dimer is: MLGFFQQPKPRKKKKKK (tpy) KK (Dye) DOTA.
The preparation method of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, the single amino acid sequence of described polypeptide polymer is: MLGFFQQPKPRKKKKKK (tpy) K (tpy) KK (Dye) DOTA.
The preparation method of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, described polypeptide polymer is polypeptide tripolymer, the polypeptide tetramer or polypeptide pentamer.
Dimeric peptide chain of the present invention there is a terpyridine moieties, can only dimer be formed; There are two terpyridine moieties in polypeptide polymer monomer of the present invention, can be formed by covalent linkage by a lot of monomers in theory, but in this application, because the reason such as sterically hindered causes the polymerization degree of described polypeptide polymer at 3-5.
The preparation method of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, described difunctional chelation group is DOTA, and described DOTA is tri-tert DOTA.
The preparation method of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, described terpyridine moieties is selected from 4'-(4-carboxymethoxyl phenyl)-2,2':6', 2''-terpyridyl), 4'-(4-carboxyl phenyl)-2,2':6', one in 2''-terpyridyl, 4'-carboxyl-2,2':6', 2''-terpyridyl.
The preparation method of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, described fluorophor sends strong fluorescence at 610nm, realizes fluoroscopic examination.
The preparation method of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of the present invention, described fluorophor is hemicyanine dye; Described fluorophor is preferably from 2-[4-(N, N-bis-propyloic) amino] styryl-1,3,3-Three methyl Benzene diindyl iodine is called for short BIDC1,2-[4-(N, N-bis-propyloic) is amino] styryl-1-butyl-3,3-dimethyl benzene diindyl iodine (BIDC2) and 2-[4-(N, N-bis-propyloic) amino] one in styryl-1-octyl group-3,3-dimethyl benzene diindyl iodine (BIDC3).
The present invention also provides a kind of described Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe to the identification of neurokinine-1 receptor protein-specific in the application of the medicine for the preparation of diagnosing, ovarian cancer, mammary cancer, prostate cancer, neurospongioma, colorectal carcinoma.
Novel Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer provided by the invention or polypeptide polymer, containing one section of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 sequence, a difunctional intercalating agent and fluorophor in monomer whose molecule, realize fluorescence, the difunctional imaging of magnetic, stable dimer or polymkeric substance can be formed by complexation of metal ions containing terpyridyl in addition.
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide monomer of the present invention is linear polypeptide.
Dimer of the present invention and polymkeric substance are film like structures.
Described Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe is red-purple liquid.
Magnetic imaging complexing metal of the present invention is Gd
3+, described magnetic imaging complexing metal Gd
3+mark described Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer and polypeptide polymer by difunctional chelation group DOTA.Described nk 1 receptor albumen is as molecular target, and polypeptide dimer and polymkeric substance realize lesion detection by this albumen of specific binding.
The monomer of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer disclosed by the invention is MLGFFQQPKPRKKKKKK (tpy) KK (Dye) DOTA (Met-Leu-Gly-Phe-Phe-Gln-Gln-Pro-Lys-Pro-Arg-Lys-Lys-Lys-Lys-Lys-Lys (tpy)-Lys-Lys (Dye)-DOTA), and structure is:
Described magnetic imaging Metal Gd
3+mark described polypeptide by a difunctional chelation group, described dimer is obtained by the polymerization of terpyridyl complexation of metal ions, and described fluorophore, under 540nm excites, sends intense fluorescence 610, and described Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer is red-purple liquid;
The monomer of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer is MLGFFQQPKPRKKKKKK (tpy) K (tpy) KK (Dye) DOTA (Met-Leu-Gly-Phe-Phe-Gln-Gln-Pro-Lys-Pro-Arg-Lys-Lys-Lys-Lys-Lys-Lys (tpy)-Lys (tpy)-Lys-Lys (Dye)-DOTA), and structure is:
Wherein M represents methionine(Met), and L represents leucine, and G represents glycine, and F represents phenylalanine, Q represents glutamine, and P represents proline(Pro), and K represents Methionin, and R represents arginine, tpy represents terpyridyl, and Dye represents a kind of fluorophore 2-[4-(N, N-bis-propyloic) is amino] styryl-1,3,3-Three methyl Benzene diindyl iodine, DOTA represents tri-tert 1,4,7,10-tetraazacyclododecanand-1,4,7,10-tetraacethyl.First the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer designed in the present invention and polymkeric substance use microwave Peptide synthesizer solid phase synthesis Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 sequence, then pass through solid phase Fmoc method improvement on synthesis monomer at reaction flask, then carry out Gd in aqueous
3+mark, finally adds Fe again
2+obtain polypeptide dimer or polypeptide polymer.
The invention has the beneficial effects as follows:
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe to the identification of neurokinine-1 receptor protein-specific provided by the invention and preparation and application thereof.(1) this probe and Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer and polymkeric substance are the parts with nk 1 receptor albumen with high-affinity, and film like structures is easier to associated proteins, improve the sensitivity detected.(2) this polypeptide dimer and polypeptide polymer by difunctional chelation group by Metal Gd
3+mark is on peptide molecule, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe in vivo through mark gathers tumor locus with nk 1 receptor protein-specific in conjunction with dense, utilize magnetic imaging and optical imagery two kinds of signal detection neurokinine-1 receptor albumen, can be used for the examination and the early diagnosis that realize tumour, identifiable design high risk population and early lesion crowd, improve detection sensitivity.
Accompanying drawing explanation
Fig. 1 is the mass spectroscopy spectrogram of described Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer monomer.
Fig. 2 is the HPLC(high performance liquid chromatography of described Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer monomer) analysis of spectra.
Fig. 3 is the mass spectroscopy spectrogram of described Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer monomer.
Fig. 4 is the HPLC(high performance liquid chromatography of described Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer monomer) analysis of spectra.
Fig. 5 is the co-focusing imaging of described Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer in HBE cell (people's normal bronchial cell) and A549 cell (human lung carcinoma cell).
Fig. 6 is the magnetic signal of described Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer in lung cancer patient serum and normal human serum.
Embodiment
In order to understand the present invention better, illustrate content of the present invention further below in conjunction with embodiment, but the content of invention is not only confined to the following examples.
In order to understand the present invention better, illustrate content of the present invention further below in conjunction with embodiment, but the content of invention is not only confined to the following examples.
Agents useful for same of the present invention and instrument:
| HPLC | GE,AKTA |
| Microwave Peptide Synthesizer | CEM |
| MALDI-TOF | AB SCIE,4800Plus |
| 0.5T MR NM2011Analyst | Shanghai Niumag Corporation |
| Confocal Laser Scanning Microscopy | Olympus |
Embodiment 1:
The preparation method of described Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer comprises the following steps:
The preparation of a, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 sequence
(1) synthesis of microwave Peptide synthesizer is used, by Rink amide resins (312mg, 0.25mmol) with Fmoc methionine(Met) (290.6mg, 0.75mmol) be placed in reaction flask, add organic bases and couplant, in DMF solution, after reacting 30min under microwave condition, remove Fmoc blocking group, obtain the resin with methionine(Met) coupling;
(2) repeating step (1) successively, wherein raw material amino acid replaces with Fmoc leucine (265.06mg, 0.75mmol) successively, Fmoc glycine (223.0mg, 0.75mmol), Fmoc phenylalanine (290.57mg, 0.75mmol), Fmoc phenylalanine (290.57mg, 0.75mmol), the related Fmoc glutamine (458.03mg, 0.75mmol) having Trt protecting group in side, the related Fmoc glutamine (458.03mg, 0.75mmol) having Trt protecting group in side, Fmoc proline(Pro) (253.03mg, 0.75mmol), the related Fmoc Methionin (351.41mg, 0.75mmol) having Boc protecting group in side, Fmoc proline(Pro) (253.03mg, 0.75mmol), the related Fmoc arginine (513.58mg, 0.75mmol) having Pbf protecting group in side, the related Fmoc Methionin (351.41mg, 0.75mmol) having Boc protecting group in side, the related Fmoc Methionin (351.41mg, 0.75mmol) having Boc protecting group in side, the related Fmoc Methionin (351.41mg, 0.75mmol) having Boc protecting group in side, the related Fmoc Methionin (351.41mg, 0.75mmol) having Boc protecting group in side, the related Fmoc Methionin (351.41mg, 0.75mmol) having Boc protecting group in side, finally obtains the resin with above-mentioned amino acid successively coupling,
(3) step (2) is obtained and the resin of above-mentioned amino acid successively coupling and the related Fmoc Methionin (339.38mg having Alloc protecting group in side, 0.75mmol), be placed in reaction flask, add organic bases and couplant, in DMF solution, after reacting 30min under microwave condition, obtain further with the resin of the Fmoc Methionin coupling with Alloc protecting group;
The preparation of b, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer
(1) in the resin of step a gained and above-mentioned amino acid successively coupling, Pd (Ph is added
3p)
4(57.78mg, 0.05mmol) and PhSiH
3(0.74mL, 6mmol), reacts 10 minutes, removes the related Allco had on the Fmoc lysine side-chain of protecting group Alloc in side in DMF.
(2) resin and terpyridyl (415.5mg, 0.75mmol) that remove Allco on Fmoc lysine side-chain are placed in reaction flask, add organic bases and couplant, in DMF solution after reaction, remove Fmoc blocking group.
(3) repeating step (2), its Raw replaces with that side is related the Fmoc Methionin (351.41mg, 0.75mmol) of protecting group Boc, the related Fmoc Methionin (339.38mg, 0.75mmol) having protecting group Allco in side successively.
(4) repeating step (1), (2), its Raw replaces with hemicyanine dye (415.5mg, 0.75mmol).
(5) repeating step (2), its Raw replaces with DOTA (429.29mg, 0.75mmol), finally obtains the resin with above-mentioned amino acid successively coupling;
(6) resin of cutting step (5) gained and above-mentioned amino acid coupling successively, obtains Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide monomer.Product is prepared through C18 preparative column.
(7) complexing Gd
3+ion, takes polypeptide monomer 8mg, is dissolved in 5mL ultrapure water, uses 0.1M NaOH to regulate pH to 6.0, adds the GdCl that concentration is 20mM
3374 μ L(3eq), pH is adjusted to 5.0, stirred overnight at room temperature.Reaction solution is adjusted PH to 7.0-8.0, stirs 3h, make excessive GdCl
3precipitation, filters.Filtrate to be dialysed 12h with 0.001MEDTA, then to dialyse 12h with ultrapure water, and freeze-drying must expect Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide monomer of mark.MALDI-TOF mass spectrometry results: m/z=3689.0.
(8) complexing metal Gd is taken
3+polypeptide monomer 8mg, be dissolved in 5mL ultrapure water, use 0.1M NaOH to regulate pH to 5.0, add the FeCl that concentration is 20mM
2340 μ L(3eq), pH is adjusted to 5.0, stirred overnight at room temperature.To dialyse 24h with ultrapure water, a water is changed in centre, and freeze-drying must expect Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer of mark is about 66mg(productive rate about 7.2%), purity is greater than 95%.
Further, described HPLC method 1 is for using GE AKTA purifier100 system disposition C18 preparative column (3cm × 25cm, 10 μm), ultraviolet 220nm and 550nm double UV check, gradient elution 50 minutes, flow velocity 17mL/min, wherein mobile phase A is ultrapure water (0.05%TFA), B is methyl alcohol (0.05%TFA); Initial concentration is 30%A and 70%B, and linear gradient leaching is to 100%B.
HPLC method 2 is GE AKTA purifier100 system disposition C18 analytical column (1cm × 15cm, 10 μm), ultraviolet 220nm and 550nm double UV check, gradient elution 40 minutes, flow velocity 1mL/min, wherein mobile phase A is ultrapure water (0.05%TFA), B is methyl alcohol (0.05%TFA); Initial concentration is 10%A and 90%B, and linear gradient leaching is to 100%B.
Embodiment 2:
The present embodiment is the improvement that the basis of embodiment 1 is carried out, part identical with embodiment 1 in the present embodiment, please refer to content disclosed in embodiment 1 and understands, and content disclosed in embodiment 1 also as the content of the present embodiment, should not do repeated description herein.
The preparation method of described Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer comprises the following steps:
The preparation of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer
(1) solid phase Fmoc technology is adopted; by the resin of step a gained in embodiment 1 and above-mentioned amino acid successively coupling and the related Fmoc Methionin (339.38mg having protecting group Allco in side; 0.75mmol) be placed in reaction flask; add organic bases and couplant, react in DMF solution and be coupled on peptide chain.
(2) in the resin of step (1) gained and above-mentioned amino acid successively coupling, Pd (Ph is added
3p)
4(105.6mg, 0.1mmol) and PhSiH
3(1.48mL, 12mmol), reacts 10 minutes, removes two related Allco had on the Fmoc lysine side-chain of protecting group Alloc in side in DMF.
(3) resin and terpyridyl (831mg, 1.5mmol) that remove Allco on Fmoc lysine side-chain are placed in reaction flask, add organic bases and couplant, in DMF solution after reaction, remove Fmoc blocking group.
(4) repeating step (3), its Raw replaces with that side is related the Fmoc Methionin (351.41mg, 0.75mmol) of protecting group Boc, the related Fmoc Methionin (339.38mg, 0.75mmol) having protecting group Allco in side successively.
(5) repeating step (2), (3), its Raw replaces with hemicyanine dye (415.5mg, 0.75mmol).
(6) repeating step (3), its Raw replaces with DOTA (429.29mg, 0.75mmol), finally obtains the resin with above-mentioned amino acid successively coupling;
(7) resin of cutting step (6) gained and above-mentioned amino acid coupling successively, obtains Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide monomer.Product is prepared through C18 preparative column.
(8) complexing Gd
3+ion, takes polypeptide monomer 8mg, is dissolved in 5mL ultrapure water, uses 0.1M NaOH to regulate pH to 6.0, adds the GdCl that concentration is 20mM
3328 μ L(3eq), pH is adjusted to 5.0, stirred overnight at room temperature.Reaction solution is adjusted PH to 7.0-8.0, stirs 3h, make excessive GdCl
3precipitation, filters.Filtrate to be dialysed 12h with 0.001MEDTA, then to dialyse 12h with ultrapure water, and freeze-drying must expect Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide monomer of mark.MALDI-TOF mass spectrometry results: m/z=4182.0.
(9) complexing metal Gd is taken
3+polypeptide monomer 8mg, be dissolved in 5mL ultrapure water, use 0.1M NaOH to regulate pH to 5.0, add the FeCl that concentration is 20mM
2298 μ L(3eq), pH is adjusted to 5.0, stirred overnight at room temperature.To dialyse 24h with ultrapure water, a water is changed in centre, and freeze-drying must expect Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer of mark is about 80mg(productive rate about 8%), purity is greater than 95%.
Further, described HPLC method 1 is for using GE AKTA purifier100 system disposition C18 preparative column (3cm × 25cm, 10 μm), ultraviolet 220nm and 550nm double UV check, gradient elution 50 minutes, flow velocity 17mL/min, wherein mobile phase A is ultrapure water (0.05%TFA), B is methyl alcohol (0.05%TFA); Initial concentration is 40%A and 60%B, and linear gradient leaching is to 100%B.
HPLC method 2 is GE AKTA purifier100 system disposition C18 analytical column (1cm × 15cm, 10 μm), ultraviolet 220nm and 550nm double UV check, gradient elution 50 minutes, flow velocity 1mL/min, wherein mobile phase A is ultrapure water (0.05%TFA), B is methyl alcohol (0.05%TFA); Initial concentration is 20%A and 80%B, linear gradient leaching 18min to 60%B, then linear gradient leaching 30min to 80%B.
Effect example 1. polypeptide polymer compares A549 cell and the fluorescence intensity in HBE cell
Cell aspect detects polypeptide polymer fluorescence in lung carcinoma cell and normal cell strong and weak.In A549 cell and HBE cell, add the polypeptide polymer that 1mL concentration is 20 μMs respectively, hatch 30min, suck Incubating Solution, wash 3 times with phosphoric acid buffer.Then cell is placed in imaging under laser confocal scanning microscope.Experiment finds, because NK-1 protein content in HBE cell walls is little, polypeptide polymer not easily enters HBE cell, and containing NK-1 albumen in A549 cell walls, thus polypeptide polymer by with NK-1 protein binding, more easily enter A549 cell.See the fluorescence intensity of the fluorescence intensity in Fig. 5, A549 cell apparently higher than HBE cell.So detect nk 1 receptor albumen by the method for fluorescence imaging thus realize the detection of tumour.
The magnetic signal of effect example 2. polypeptide polymer in lung cancer patient serum compares with the magnetic signal in normal human serum
Set up clinical detection lung cancer model.Add that the magnetic signal after polypeptide polymer is different to be diagnosed by comparing lung cancer patient and the serum of normal people.Choose 15 routine lung cancer patients and 15 routine normal human serums respectively, add polypeptide polymer and detect its T
1value, each sample in triplicate.Experiment finds, due in the serum of nonsmall-cell lung cancer patient containing NK-1 albumen, therefore with polypeptide in conjunction with time its magnetic signal can strengthen, show as T
1value diminishes, see Fig. 6, and the T of the serum of 15 routine lung cancer patients
1value is all lower than the T of normal human serum
1value.Take 810ms as the T of boundary, patients serum
1value is all lower than 810ms, and minority can be low to moderate 700ms, and because not containing nk 1 receptor albumen, its T in normal human serum
1value is all higher than 810ms.Illustrate that Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer has the susceptibility of higher detection neurokinine-1 receptor albumen, there are the potentiality for kinds of tumors diagnosis, and the method is sensitive, simple to operate.
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe to the identification of neurokinine-1 receptor protein-specific provided by the invention and preparation and application thereof.(1) this probe and Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer and polymkeric substance are the parts with nk 1 receptor albumen with high-affinity, and film like structures is easier to associated proteins, improve the sensitivity detected.(2) this polypeptide dimer and polypeptide polymer by difunctional chelation group by Metal Gd
3+mark is on peptide molecule, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe in vivo through mark gathers tumor locus with nk 1 receptor protein-specific in conjunction with dense, utilize magnetic imaging and optical imagery two kinds of signal detection neurokinine-1 receptor albumen, can be used for the examination and the early diagnosis that realize tumour, identifiable design high risk population and early lesion crowd, improve detection sensitivity.
Claims (10)
1. the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe of pair neurokinine-1 receptor protein-specific identification, it is characterized in that: described Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe is polypeptide dimer or polypeptide polymer, monomer whose is formed by connecting by Methionin by one section of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 aminoacid sequence MLGFFQQPKPR, difunctional chelation group, fluorophor and terpyridine moieties.
2. Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe according to claim 1, is characterized in that: the single amino acid sequence of described polypeptide dimer is: MLGFFQQPKPRKKKKKK (tpy) KK (Dye) DOTA; The single amino acid sequence of described polypeptide polymer is: MLGFFQQPKPRKKKKKK (tpy) K (tpy) KK (Dye) DOTA.
3. Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe according to claim 1, is characterized in that: the polymerization degree of described polypeptide polymer is 3-5.
4. Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe according to claim 1, is characterized in that: described difunctional chelation group is tri-tert DOTA; Described difunctional chelation group can chelated mineral Gd
3+, F
19or Mn
2+.
5. Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe according to claim 1, it is characterized in that: described terpyridine moieties is selected from 4'-(4-carboxymethoxyl phenyl)-2,2':6', 2''-terpyridyl, 4'-(4-carboxyl phenyl)-2,2':6', 2''-terpyridyl, 4'-carboxyl-2,2':6', 2''-terpyridyl.
6. Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe according to claim 1, is characterized in that: described fluorophor is hemicyanine dye; Described fluorophor is preferably from 2-[4-(N, N-bis-propyloic) amino] styryl-1,3,3-trimethylammonium-1H-benzindole iodine, 2-[4-(N, N-bis-propyloic) is amino] styryl-1-butyl-3,3-dimethyl-1H-benzindole iodine and 2-[4-(N, N-bis-propyloic) amino] styryl-1-octyl group-3,3-dimethyl-1H-benzindole iodine.
7. prepare a method for the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe described in claim 1-6, it is characterized in that: said method comprising the steps of:
The preparation of a, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 sequence
(1) Rink amide resins and Fmoc methionine(Met) are placed in reaction flask, add organic bases and couplant, in DMF solution, after reacting 30min under microwave condition, then remove Fmoc blocking group, obtain the resin with methionine(Met) coupling;
(2) repeating step (1) successively, wherein raw material amino acid replaces with Fmoc leucine successively, Fmoc glycine, Fmoc phenylalanine, Fmoc phenylalanine, the related Fmoc glutamine having protecting group in side, the related Fmoc glutamine having protecting group in side, Fmoc proline(Pro), the related Fmoc Methionin having protecting group in side, Fmoc proline(Pro), the related Fmoc arginine having protecting group in side, the related Fmoc Methionin having protecting group in side, the related Fmoc Methionin having protecting group in side, the related Fmoc Methionin having protecting group in side, the related Fmoc Methionin having protecting group in side, the related Fmoc Methionin having protecting group in side, finally obtain the resin with above-mentioned amino acid successively coupling,
(3) step (2) is obtained and the related Fmoc Methionin having Alloc protecting group of the resin of above-mentioned amino acid successively coupling and side, be placed in reaction flask, add organic bases and couplant, in DMF solution, after reacting 30min under microwave condition, obtain further with the resin of the Fmoc Methionin coupling with Alloc protecting group;
The preparation of b, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer
(1) in the resin of step a gained and amino acid successively coupling, Pd (Ph is added
3p)
4and PhSiH
3, react 10 minutes in DMF, remove the Allco protecting group on lysine side-chain;
(2) step (1) products therefrom and terpyridyl are placed in reaction flask, add organic bases and couplant, in DMF solution after reaction, then remove Fmoc blocking group;
(3) then under the condition of organic bases, couplant and DMF solution, successively with side the related Boc of having protecting group Fmoc Methionin, side is related has the Fmoc Methionin of Allco protecting group to react;
(4) repeating step (1), (2), its (2) Raw replaces with hemicyanine dye, DOTA, finally obtain being connected with terpyridyl, hemicyanine dye, DOTA with the resin of amino acid successively coupling;
(5) resin of cutting step (4) gained and amino acid coupling successively, obtains Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer monomer;
(6) Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer monomer is dissolved in ultrapure water, adds GdCl
3, after reaction treatment, obtain Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer monomer of mark;
(7) by Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer monomer of mark is dissolved in ultrapure water, adds FeCl
2, reaction, aftertreatment obtains Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide dimer of mark;
The preparation of c, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer
(1) by the resin of step a gained and amino acid successively coupling, remove Fmoc blocking group, under organic bases, couplant and DMF solution existent condition, have the Fmoc Methionin of protecting group Allco to react with side is related, Fmoc Methionin is coupled on peptide chain;
(2) in step (1) products obtained therefrom, Pd (Ph is added
3p)
4and PhSiH
3, react 10 minutes in DMF, remove the Allco protecting group on two Fmoc lysine side-chains;
(3) step (2) products obtained therefrom and terpyridyl are placed in reaction flask, add organic bases and couplant, in DMF solution after reaction, remove Fmoc blocking group;
(4) repeating step (3), its Raw replaces with Fmoc Methionin, the related Fmoc Methionin having Allco protecting group in side of the side related Boc of having protecting group successively;
(5) repeating step (2), (3), wherein step (3) Raw replaces with hemicyanine dye, DOTA successively, finally obtain being connected with terpyridyl, hemicyanine dye, DOTA with the resin of amino acid successively coupling;
(6) resin of cutting step (5) gained and amino acid coupling successively, obtains Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer monomer;
(7) Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer monomer is dissolved in ultrapure water, adds GdCl
3, reaction, aftertreatment obtains Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer monomer of mark;
(8) by Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer monomer of mark is dissolved in ultrapure water, adds FeCl
2, reaction, aftertreatment obtains Gd
3+the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide polymer of mark.
8. the preparation method of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe according to claim 7, is characterized in that: described side is related the Fmoc glutamine of protecting group, side is related the Fmoc Methionin of protecting group, related Fmoc glutamine, the Fmoc Methionin of the side related Boc of having protecting group, the related Fmoc arginine having Pbf protecting group in side having the Fmoc arginine of protecting group to be followed successively by the side related Trt of having protecting group in side.
9. the preparation method of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe according to claim 7, is characterized in that: described organic bases is DIPEA, and described couplant is phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl.
10. the application of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 polypeptide probe on the medicine for the preparation of diagnosing, ovarian cancer, mammary cancer, prostate cancer, neurospongioma, colorectal carcinoma described in a claim 1-9.
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| CN1738652A (en) * | 2003-01-13 | 2006-02-22 | 伯拉考成像股份公司 | Improved linkers for pharmaceutical compounds |
| CN1874792A (en) * | 2003-09-03 | 2006-12-06 | 布里斯托尔-迈尔斯.斯奎布制药公司 | Compounds containing matrix metalloproteinase substrates and methods of their use |
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| CN1738652A (en) * | 2003-01-13 | 2006-02-22 | 伯拉考成像股份公司 | Improved linkers for pharmaceutical compounds |
| CN1874792A (en) * | 2003-09-03 | 2006-12-06 | 布里斯托尔-迈尔斯.斯奎布制药公司 | Compounds containing matrix metalloproteinase substrates and methods of their use |
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